scholarly journals Cytoplasmic p53 polypeptide is associated with ribosomes.

1997 ◽  
Vol 17 (6) ◽  
pp. 3146-3154 ◽  
Author(s):  
B M Fontoura ◽  
C A Atienza ◽  
E A Sorokina ◽  
T Morimoto ◽  
R B Carroll
Keyword(s):  
Sodium Dodecyl ◽  
Proteinase K ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Mcf7 Cells ◽  
Gapdh Mrna ◽  
5.8S Rrna ◽  
Proteinase K Treatment ◽  
Covalently Linked

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.

p53 is covalently linked to 5.8S rRNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5145-5151
Author(s):  
B M Fontoura ◽  
E A Sorokina ◽  
E David ◽  
R B Carroll
Keyword(s):  
P53 Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Suppressor Gene ◽  
Proteinase K ◽  
Isolation And Identification ◽  
5.8S Rrna ◽  
Sds Page ◽  
Antisense Sequence ◽  
Covalently Linked

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.

scholarly journals p53 is covalently linked to 5.8S rRNA.

1992 ◽  
Vol 12 (11) ◽  
pp. 5145-5151 ◽  
Author(s):  
B M Fontoura ◽  
E A Sorokina ◽  
E David ◽  
R B Carroll
Keyword(s):  
P53 Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Suppressor Gene ◽  
Proteinase K ◽  
Isolation And Identification ◽  
5.8S Rrna ◽  
Sds Page ◽  
Antisense Sequence ◽  
Covalently Linked

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.

scholarly journals A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

2021 ◽  
Author(s):  
Mo Chen ◽  
Suyong Choi ◽  
Tianmu Wen ◽  
Changliang Chen ◽  
Narendra Thapa ◽  
...  
Keyword(s):  
Genotoxic Stress ◽  
Akt Pathway ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Akt Activation ◽  
Pi3k Inhibitors ◽  
Phosphoinositide 3 Kinase ◽  
Induced Apoptosis ◽  
Dependent Mechanism

The tumor suppressor p53 and the phosphoinositide 3-kinase (PI3K)-Akt pathway have fundamental roles in regulating cell growth, apoptosis and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear Akt activation by a p53-dependent mechanism that is independent from the canonical membrane-localized PI3K-Akt pathway. Upon genotoxic stress a nuclear p53-PI3,4,5P3 complex is generated in regions devoid of membranes by a nuclear PI3K, and this complex recruits all the kinases required to activate Akt and phosphorylate FOXOs, inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear Akt in an on/off fashion upon stress, whereas mutant p53 stimulates high basal Akt activity, indicating a fundamental difference. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, underscoring its therapeutic relevance.

scholarly journals The Janus Face of p53-Targeting Ubiquitin Ligases

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1656 ◽  
Author(s):  
Qian Hao ◽  
Yajie Chen ◽  
Xiang Zhou
Keyword(s):  
Cancer Progression ◽  
Ubiquitin Ligases ◽  
Tp53 Gene ◽  
Mutant P53 ◽  
E3 Ubiquitin Ligases ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Cell Growth Arrest ◽  
Cancer Cell Survival ◽  
P53 Status

The tumor suppressor p53 prevents tumorigenesis and cancer progression by maintaining genomic stability and inducing cell growth arrest and apoptosis. Because of the extremely detrimental nature of wild-type p53, cancer cells usually mutate the TP53 gene in favor of their survival and propagation. Some of the mutant p53 proteins not only lose the wild-type activity, but also acquire oncogenic function, namely “gain-of-function”, to promote cancer development. Growing evidence has revealed that various E3 ubiquitin ligases are able to target both wild-type and mutant p53 for degradation or inactivation, and thus play divergent roles leading to cancer cell survival or death in the context of different p53 status. In this essay, we reviewed the recent progress in our understanding of the p53-targeting E3 ubiquitin ligases, and discussed the potential clinical implications of these E3 ubiquitin ligases in cancer therapy.

scholarly journals Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

2021 ◽  
Author(s):  
Ashley S Denney ◽  
Andrew D Weems ◽  
Michael A McMurray
Keyword(s):  
Three Dimensional ◽  
Temperature Sensitive ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Hsp90 Inhibition ◽  
New Approach ◽  
Type Function ◽  
Chaperone Interactions

Abstract Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.

scholarly journals A Protease-Resistant Catalase, KatA, Released upon Cell Lysis during Stationary Phase Is Essential for Aerobic Survival of a Pseudomonas aeruginosa oxyR Mutant at Low Cell Densities

2000 ◽  
Vol 182 (16) ◽  
pp. 4557-4563 ◽  
Author(s):  
Daniel J. Hassett ◽  
Eyad Alsabbagh ◽  
Kislay Parvatiyar ◽  
Michael L. Howell ◽  
Robert W. Wilmott ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sodium Dodecyl ◽  
Luria Broth ◽  
Serial Dilution ◽  
Cathepsin G ◽  
Proteinase K ◽  
Gene Products ◽  
Wild Type ◽  
Cell Densities ◽  
Do So

ABSTRACT A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H2O2, despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB,ahpB, and ahpCF could not restore both the serial dilution defect and H2O2 resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB andahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate ∼1.2 μM H2O2 min−1via autoxidation, a level sufficient to kill serially dilutedoxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR rendersP. aeruginosa exquisitely sensitive to both H2O2 and aminoglycosides, which are clinically and environmentally important antimicrobials.

scholarly journals Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

1991 ◽  
Vol 11 (1) ◽  
pp. 12-19 ◽  
Author(s):  
J Milner ◽  
E A Medcalf ◽  
A C Cook
Keyword(s):  
Tumor Progression ◽  
Carboxyl Terminus ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Dominant Effect ◽  
Type Mutant ◽  
Wild Type P53

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

scholarly journals Tumor suppressor p53: analysis of wild-type and mutant p53 complexes

1991 ◽  
Vol 11 (1) ◽  
pp. 12-19 ◽  
Author(s):  
J Milner ◽  
E A Medcalf ◽  
A C Cook
Keyword(s):  
Tumor Progression ◽  
Carboxyl Terminus ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Dominant Effect ◽  
Type Mutant ◽  
Wild Type P53

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

Sign in / Sign up

Export Citation Format

Share Document